The present invention lies in the field of the treatment of conditions, pathological or not, related to the accumulation and/or persistence in cells of prenylated proteins.
The nucleus of eukaryotic cells is delimited by a double membrane with pores in it, the nuclear envelope, which controls the molecular exchanges between the two nuclear and cytoplasmic compartments. This envelope partially isolates the content of the nucleus, that is to say the genetic material and all the enzymatic machinery necessary for the functions of the nuclear genome.
The nucleus envelope consists of two concentric membranes, the external membrane, in continuity with the endoplasmic reticulum, and the internal membrane. The latter is bordered on its internal face by a dense fibrillar mesh called the nuclear lamina. This is a protein lattice composed essentially of lamin polymers and associated proteins. In vertebrates, there are two sub-classes of lamins: type A lamins (lamins A and C), and type B lamins (lamins B1, B2 and B3), which all participate in the production of the lamina. The latter is held in place by association with other proteins, fixed to the internal membrane of the nuclear envelope (for journal, Gruenbaum et al 2005).
Lamins are proteins in the form of filaments belonging to the family of intermediate filaments (type V), which all have a common structure: a short N-terminal globular segment (the head) separated from another C-terminal globular segment (the tail) by a long central domain organised in several alpha helixes (the rod domain). The globular tail contains in particular a nuclear location signal (NLS) allowing addressing of the nucleus after synthesis. The central domain allows the association of two parallel lamin molecules and their organisation in filaments by the association of dimers in opposite orientations. This structure confers very resistant mechanical properties on them.
Only the A-type lamin and the B-type lamins undergo maturation after the synthesis of a precursor (Gruenbaum et al 2000). The C-type lamin is directly synthesised in its mature form.
The precursor of the A-type lamin and the B-type lamins terminates in a chracteristic CaaX unit (C is a cystein, a an amino acid with a non-charged aliphatic chain and X any amino acid, here a methionine, Levy & Cad 2003).
The C-terminal CaaX unit allows the fixing of a fatty acid (in general a C15 fatty acid, farnesyl) by virtue of a farnesyl-transferase. This prenylation (the farnesyl unit derives from a C5 base aliphatic unit called isoprenoid) enables the prelamins to be inserted in the membrane of the endoplasmic reticulum after their synthesis in cytosol. They undergo therein the action of an endoprotease, itself inserted in the envelope membrane of the reticulum and whose active site is cytosolic. The specific endoprotease of the prelamin A is Face1 (or ZMPSTE24 (Zinc Metallo-Protease homologue of yeast STE24), while Face2 (or Rce1, Ras-converting enzyme) is specific to the B prelamins. These enzymes catalyse the hydrolysis of the peptide bond between the cysteine and the following amino acid (aliphatic), shortening the prelamins by 3 amino acids. The carboxyl end of the farnesylated cysteine is then recognised by an isoprenylcysteine-carboxymethyl transferase (ICMT), which fixes a methyl group thereto by esterification.
Only the maturation of the prelamin A continues with a second endoproteolytic cleaving by Face1, which releases a farnesyl-peptide of 15 amino acids and a mature lamin A. This lamin A, which no longer includes the fatty acid, becomes soluble, and is imported into the nuclear by virtue of its nuclear location signal, and is located in the nuclear lamina itself as well in the rest of the nucleus compartment, constituting a veritable nuclear skeleton (Gruenbaum et al 2005). The on the other hand mature B lamin still has at the C-terminal end its farnesylated and methylesterified cysteine. It therefore remains inserted in the envelope membrane of the reticulum, and then in the nucleoplasmic face of the nuclear envelope, and has its location exclusive to the nuclear lamina, and the internal membrane of the nuclear envelope where it is anchored.
The term prenylation should be taken to mean the fixing to the thiol group of a cysteine either of a farnesyl chain of 15 carbon atoms, and farnesylation is then spoken of, or a geranylgeranyl chain of 20 carbon atoms, and geranylgeranylation is then spoken of (Reid et al 2004), or of any other derivative of isoprene.
The farnesylation, catalysed by the farnesyl-transferase (FTase), which recognises the C-terminal consensus sequence (CaaX), preferentially fixes a farnesyl group to the cysteine residue of the unit.
Gernaylgeranylation is the fixing by geranylgeranyl-transferase (GGTase) of a geranylgeranyl group to the cysteine residue of the unit.
These fatty acids result from the biosynthesis method which, using hydroxymethyl-glutaryl-Coenzyme A, is used by the cells for manufacturing in particular cholesterol, steroids, the haem of haemoglobin and ubiquinones (Hampton et al 1996).
The family of prenylated proteins comprises approximately 300 members in the human genome, the majority of which can be identified by the C-terminal unit CaaX (Reid et al 2004). The proteins of the families Ras, Rho, Rab (Leung et al 2006), certain proteins fulfilling an import function to the mitochondria (HDJ2), some centromeric proteins (CENPE, CENPF) are in particular prenylated (Winter-Vann & Casey 2005). In general, if in the CaaX unit, X is a serine, a methionine, a cysteine, an alanine or a glutamate, the preferentially grafted isoprenoid is farnesyl. If X is a leucine, the CaaL unit will preferably recognised by the GGTase, which will catalyse the transfer of a geranylgeranyl group (Basso et al 2006). It is probable that other groups derived from isoprene can also be fixed to this cysteine, although this is not described in the literature.
In humans, there exist three lamin genes. The LNMA gene, situated at 1q21.2-q21.3 (Wydner et al 1996), gives the lamins A and C by alternating splicing. The LMNA gene is composed of 12 exons. The start of exon 1 codes the N-terminal globular end common to lamins A and C; the end of exon 1 and up to the start of exon 7 code the central helical part; finally, the other exons code the C-terminal globular end (Levy & Cau 2003).
In fact, the gene codes for 4 differently spliced products, the C lamins and the prelamin A of which are the two main ones (Lin & Worman 1993). The differential production of lamins A and C is done by the use of an alternative splicing site at exon 10 of the pre-messenger, so that the lamin C is coded by exons from 1 to 10 and the lamin A is coded by exons from 1 to 9, the first 90 pairs of bases of exon 10, and exons 11 and 12 (A-specific lamin).
Consequently the prelamin A and the lamin C peptides are identical at the first 566 amino acids, the C-terminal ends of the C lamins and the prelamin A next containing respectively 6 and 98 specific amino acids.
The type-B lamins comprise three different proteins (Shelton et al 1981): lamins B1, B2 (the two most represented isoforms) and B3. The LMNB1 gene is situated at 5q23.3-q31.1 and comprises 11 exons coding the lamin B1 (Lin & Worman 1995). The LMNB2 gene is located at 19p13.3 and codes for the lamins B2 and B3 by an alternating splicing mechanism (Biamonti et al 1992).
The type-B lamins are expressed constituently in all the cells as from the first development stages, while the type A lamins are in general absent in the embryonic strain cells (Rober et al 1989, Stewart et al 1987) and are expressed in all the differentiated somatic cells. Their expression is subject to regulations according to the tissue and during life (Duque et al 2006). It seems that their expression is not necessary, since mice in which the lamin A expression has been specifically blocked, but which all the same express the lamin C and the other lamins, do not have any apparent phenotype (Fong et al 2006).
The lamins interact with a very high number of protein partners having very diverse functions; they are consequently involved in a large number of nuclear processes, including DNA replication and repair, control of transcription and splicing, organisation of the chromatin structure (for journal, see Shumaker et al 2003, Zastrow et al 2004, Hutchison et al 2004, Gruenbaum et al 2005). Alterations to the structure of the lamina give rise to numerous human hereditary pathologies. They are due to mutations of the genes coding the lamins, or other proteins of the lamina. These pathologies have been grouped together under the generic term laminopathies (Broers et al 2006, Mattout et al 2006). Recently, mutations in the genes of the enzymes responsible for the maturation of lamins (Facet in particular) have been identified, giving rise to pathologies also belonging to the group of laminopathies (Navarro et al 2004 and 2005).
At the present time, the only pathology in humans associated with mutations of the LMNB1 or 2 genes is a leucodystrophy caused by a complete duplication of the LMNB1 gene (Padiath et al 2006). A doubt remains on the potential implication of variation sequences found in LMNB2 in patients suffering from Barraquer-Simon syndrome (Hegele et al 2006). However, it has been demonstrated in vitro by RNAi (RNA-interference) experiments (Harborth et al 2001), and in the murin model (Vergnes et al 2004), that type B lamins are essential for cell development and integrity. This is because a lamin B1 deficiency causes perinatal mortality in mice. Moreover, the nuclei of the embryonic fibroblasts of the same LMNB1 deficient mice show remarkable alterations in the nucleus morphology, close to those observed in patients carrying mutations of the LMNA gene. In addition, it has been shown recently that B-type lamins are necessary to the formation of the division spindle during mitosis, which tends to prove that their role is dynamic and multiple during the cell cycle, not only restricted to maintenance of the architecture of the nucleus (Tsai et al 2006). On this latter role, a recent article demonstrates the structural function of the B-type lamins: cells artificially deprived of B1-type lamins have a “floating” nucleus in the cell, which turns on itself (Li et al 2007). The functional redundancy existing between the two lamins B1 and B2 is no doubt also a direct reflection of their indispensability, exerting a high selection pressure and masking the effect of any mutations of the sequence of corresponding genes.
The functional alterations in the A/C lamins, due to mutations of the LMNA gene, give rise to at least 15 disorders including very diverse pathologies in a clinical spectrum ranging from mild forms, affecting a tissue in an isolated fashion, to systemic forms that are fatal in the perinatal period.
Many mutations of the LMNA gene appreciably modify the assembly of proteins in the nuclear envelope and disturb their functioning. In the cells of various tissues, the morphology of the nuclei is altered: they often have hernias that extrude genetic material in the cytoplasm (Goldman et al 2004).
The proteins normally associated with the nuclear envelope, the B lamins, certain proteins of the nuclear pores and the LAP2 proteins are absent from the periphery of these hernias.
These morphological anomalies are followed by functional alterations, which end up by causing cell deaths. Among all the pathologies grouped together under the term laminopathies, only those relating to the abnormal accumulation of a prenylated form of protein relate to the present invention.
These are mainly the Hutchinson-Gilford syndrome, or Progeria (De Sandre-Giovannoli et at 2003, Eriksson et al 2003), and restricted dermopathy (Navarro et al 2004). In these 2 syndromes, the physiopathological cause is an accumulation and persistence in the cells of the patients of non-matured farnesylated prelamin A.
Restrictive dermopathy, fatal around the natal period, is characterised by clinical signs that are almost all the consequence of a cutaneous deficit that restricts movements in utero. This pathology is very rare. The skin is rigid and tensioned, and yields in places, causing for example tears at the armpits or neck. The eyelashes, eyebrows and skin hair are absent or very sparse. Hydramnios is often present, and a reduction in fetal movements is signaled as from the sixth month of pregnancy. At the skeletal level, radiography reveals contractions at all of the joints, deformed feet, thin, dysplastic and bi-partited clavicles, ribbon-shaped ribs, tubular long bones of the arms and demineralisation at the cranium. Transmission of fatal restrictive dermopathy is autosomal recessive.
LMNA and ZMPSTE24/Face1 mutations have been reported for this pathology (Navarro et al 2004). In both cases, the physiopathological mechanism is the same: the prelamin A cannot mature (zero Face1 mutation or disappearance of the cleavage site by mutation of the prelamin A), and remains farnesylated, and therefore inserted in the nuclear membrane. The accumulation and persistence in the cells of these abnormal precursors, which probably prevent normal interaction of the lamins B and C with their partners, causes death of the cells and, very soon, of the patient. It has been clearly demonstrated that it is indeed the persistence of the farnesylated group rather than the absence of mature lamin A, as might have been thought at first, that is responsible for the cell toxicity (Fong et al 2004).
In April 2003, from cross-checking the symptoms common to acromandibular dysplasia and some diseases resulting in premature aging, the inventors showed that Progeria, the most typical and most serious form of premature aging, results from a mutation of the LMNA gene (De Sandre-Giovannoli et al 2003). Children afflicted by this illness, also referred to as Hutchinson-Gilford syndrome, suffer from accelerated aging, up to ten times more rapid than that of a normal individual, and have a life expectancy that does not exceed 13 years. In France, one child out of approximately six million is affected. The symptoms are cutaneous aging, baldness, reduction in the size of the jaw and problems related to old age, for example stiffness in the joints and cardiovascular disorders. The latter, such as myocardial infarction or atherosclerosis, are often the cause of death.
The mutation involved, situated at exon 11 of the LMNA gene, activates a cryptic splicing site of the pre-mRNA, leading to a deleted mRNA of 150 nucleotides (De Sandre-Giovannoli et at 2003, Eriksson et al 2003). This deleted mRNA is translated into an abnormal prelamin A, progerin, which cannot be matured into normal lamin A: the absence of 50 amino acids of exon 11 comprising the protease recognition site blocks the second cleavage of the progerin, the C-terminal end of which keeps its farnesylated group. It therefore remains inserted in the nucleoplasmic face of the nuclear envelope, which has characteristic alterations, hernias of the nucleoplasm in the cytosol and abnormalities in the distribution of the peripheral heterochromatin (Goldman et al 2004). Here also, it is the persistence of the farnesylated group, moreover necessary for anchoring to the envelope membrane of the reticulum in which there are located some of the enzymes responsible for maturation (cleavages, methylation) which is responsible for the cell toxicity of the progerin (Fong et al 2004).
These systemic pathologies have the particularity of being associated with the premature appearance of signs normally related to aging. Their common physiopathological characteristic is to generate a prenylated lamin, with the consequences described.
Two recent studies have shown that a reduction in the intranuclear accumulation of the farnesylated prelamin, truncated or not, effectively prevents the appearance of the cell phenotype. The first was carried out on the progeroid murin model deficient in Face1 protease (Pendas et al 2002). When they are crossed with mice expressing half the amount of lamin A (LMNA+/−mouse), the effects of the absence of Face1 are less (Varela et al 2005). The second study shows that the treatment of cells of HGPS patients with morpholino (antisens oligonucleotides) targeting the cryptic splicing site does away with the mutant phenotype (Scaffidi & Misteli 2005).
Several recent studies (Scaffidi & Misteli 2006, Cao et al 2007) show the involvement of lamin A in the physiological aging process. In particular, it has been demonstrated that, during physiological aging, an aberrant lamin A accumulates over time at the periphery of the cell nucleus. This aberrant lamin is fact progerin, the cell, during its normal life and functioning accidently using from time to time the cryptic splicing site of exon 11, the progerin produced accumulates little by little under the lamina. Finally, the “normal” aged cell may present hernias characteristic of a laminopathy caused by these accidental splicing events, which cause its death.
It appears that identical molecular mechanisms are firstly responsible for the signs of premature aging in individuals suffering from Progeria and secondly, at a much lower level, are involved in the physiological aging of individuals not carrying mutations.
There exist in the prior art two therapeutic approaches described for improving the cell phenotype caused by the pathological production of progerin. The first of these solutions is quite simply to prevent the use by the spliceosome of this cryptic splicing site in exon 11, by “masking” it by treatment with an antisens oligonucleotide (Scaffidi & Mistelli 2005), or with a retrovirus producing an siRNA (Huang et al 2005). The results are promising in vitro, but it is a case here of “gene” therapy, and the development of a medication around this approach is necessarily long and complicated, with all the drawbacks related to the vectorisation of the OASs in order to obtain an in vivo effect. The second solution consists of inhibiting the farnesyl-transferase, the enzyme that catalyses the transfer of the farnesyl group on the prelamins from farnesyl-pyrophosphate. When such inhibitors (FTI) are used, a “normal” nuclear envelope is only partially restored on HGPS cells (Progeria) in culture, and the survival of RD mice (KO ZMPSTE24) is improved (Glynn & Glover 2005, Capell et al 2005, Toth et at 2005, Fong et al 2006).
However, blocking and farnesylation may cause a compensatory geranylgeranylation (Bishop et al 2003).
In addition, it has been reported recently that FTIs cause a stoppage of the cell cycle by blocking the proteasome (Demyanets et al 2006, Efuet & Keyomarsi 2006). Thus the treatment no doubt causes an accumulation in the nucleoplasm of progerin probably ubiquitinylated not degraded by the proteasome.
In addition, recent works report that the reduction in the level of farnesylation of the progerin in vivo is very low, around 5% (Young et al 2006), which does not suffice to explain the restoration in the nuclear morphology observed in vitro.
Finally, the FTIs are specific to only one of the protein prenylation routes and cannot be envisaged as global inhibitors of post-translation prenylations.
Moreover, it is reported that the total absence of one of the enzymes of this route, mevalonate-kinase, is fatal during infancy (homozygote mutation loss of function of the gene coding for this enzyme, a syndrome reported by Hoffmann et al 2003).